The present invention relates to the animal cell medicine, specifically to a microencapsulated medicine of ox adrenal medulla pheochromocyte (BCC) for treating pain.
Pain is a common symptom caused by several disease factors and causing serious soreness to patients. Although current medicines, such as morphine-like, can bring analgesic effects in short-term, its repeating administration usually result in drug-resistance and habituation. As adrenal medulla pheochromocyte can secrete certain substance in association with analgesic effects, such as methionine enkephalin(MEK), leucineenkephalin (LENK) and monamine substances, so once pheochromocyte had been implanted into subarachnoid space of spinal of human or animal, it can act as a xe2x80x9cmini bio-pumpxe2x80x9d and release analgesic substances continuously in a long period, and wouldn""t result in drug-resistance and habituation. So in the beginning of 1980""s, two research groups from USA and Switzerland tried to implant homogenous (human, rat) adrenal medulla tissues and chromaffin cells into subarchnoid space of spinal cord for treating pain and had finally obtained satisfactory results. Because of the less sources of human adrenal medulla tissues and chromaffin cells, in 1990""s, the research group from USA had tried to implant heterogeneous ox adrena medulla pheochromocyte (hereafter abbreviated as BCC) into subarachnoid space of spinal cord of cancer patients to cure pain. In order to overcome the immuneexclusion reaction, they used polyacrylamide hollow fiber tube of 5 cm in length and 1 mm in diameter to coat BCC. As the said hollow fiber tube only allow small molecules to pass through, the secreta of BCC can diffused slowly and uniformly from the fiber tube to perform the analgesic effect, while macromolecular immunoglobulin in host body can""t pass through the hole of tube wall, so that the cells can survive in host body in a long period(about 1 year) and allows continuous delivery of analgesic substances to relief the pain of patients. But the hollow fiber tube has a large volume. On one side, the dead volume of tube affect the dispersion of nutrients and metabolites, which make the cells inside tube can""t survive chronically. On the other hand, implantation of fiber tube with large volume into subarachnoid space of spinal cord would stimulate and oppress spinoneure, thereby cause many undesirable side effects. Moreover, as the volume of hollow fiber tube is large, it must to be implanted into subarachnoid space by surgical operation, which would injure patient more or less. In the meantime, the hollow fiber tube made of materials such as chitosan, polyacrylamide and carboxymethyl cellulose (used in USA) have poor tissue biocompatibility, which cause tissue reaction in host body. On the other side, the microcapsules with three layer membrane structure of sodium alginatepolylysine-sodium alginate (APA microcapsule) have little volume (200-1000 xcexcm in diameter) and high biocompatibility, which contribute that microcapsule survive chronically and well in host cell and the cells inside microcapsule survive chronically. The experiments have indicated that microcapsule membrane can cut off macromolecule with molecular weight beyond 110,000 Kd (dalton), prevent immunoglobulin and immunological competence cell pass through the said membrane into microcapsule to destroy inside animal cell, which thus provide proper immunity protection. Experiments also indicated that APA microcapsules have proper biocompatibility, and have long term existence (about one year and half) in small or big animal body. The research on implantation of islands of Langerhans, liver cells, parathyroid and genetic recombinant growth-hormone secretory cells for treating disease model animal have provided the evidence that the said APA microcapsule protect heterogeneous implant from host immune system. However, so far, there is no report about using APA microencapsulated pheochromocyte of ox adrenal medulla as medicine for curing pain.
The object of the present invention is to provide APA microencapsulated medicine of ox adrenal medulla pheochromocyte for curing pain, which have advantages of proper biocompatibility, long acting time, lower side effect and can operate easily.
The present inventor has conducted deep research based on aforementioned existing technique, found that employing following embodiment can arrive at said aims, thus accomplish the present invention.
Embodiment of the present invention described as follows:
1. Microencapsulated medicine of ox adrenal medulla pheochromocyte for treating pain, which is prepared through the following steps:
(1) mixing the ox adrenal medulla pheochromocyte with 10-20 g/L solution of sodium alginate to form suspension, the said suspension contains 0.1-1xc3x971010 cells per liter.
(2) dispersing the suspension of step (1) into 80-120 mmol/L calcium chloride solution or calcium lactate solution, in the form of microdrop of diameter of 150-1000 xcexcm by spray device, the proportion of the two solution is to let 1 liter mixture contains 0.1-1xc3x97108 cells, setting 5-20 minutes, removing the supernatant after depositing completely, obtaining the calcium alginate bead deposit which contains ox adrenal medulla pheochromocyte;
(3) adding the deposit derived from step (2) into the 0.3-0.7 g/L polylysine solution, the proportion of the two solution is to let 1 liter mixture contains 0.2-2xc3x97108 cells, mixing it uniformly, setting 5-20 minutes, removing the supernatant after deposit completely, getting the deposit;
(4) adding the deposit derived from step (3) into the 1.0-2.0 g/L sodium alginate solution, the proportion of the two solution is to let 1 liter mixture contains 0.2-2xc3x97108 cells, mixing it uniformly, setting 3-15 minutes, removing the supernatant after deposit completely, getting the deposit;
(5) adding the deposit derived from step (4) into 40-70 mmol/L sodium citrate solution, the proportion of the two solution is to let 1 liter mixture contains 0.2-2xc3x97108 cells, mixing it uniformly, setting 5-20 minutes, removing the supernatant after deposit completely, obtaining the microencapsulated medicine of ox adrenal medulla pheochromocyte deposit;
(6) washing the deposit derived from step (5) by adding it into the 9 g/L sodium chloride solution, finally transferring the deposit into a cell culture, and storing it as microencapsulated medicine of ox adrenal medulla pheochromocyte.
2. The microencapsulated medicine of ox adrenal medulla pheochromocyte in item 1, wherein the said ox adrenal medulla pheochromocyte has a purity of at least 80%.
3. The microencapsulated medicine of ox adrenal medulla pheochromocyte in item 1, which is characterized in that in said step (2) dispersing the suspension derived from step (1) in microdrop state with a diameter of 180-500 xcexcm.
As for use, the APA-microencapsulated BCC (2xcx9c9xc3x97106 cells) was injected into subarachnoid space of spinal cord of patients (e.g. cancer patient),which would cause analgesic effect within 4-24 hours and the effect could last for over 9 months by each injection.
The detailed explanation of embodiments of the invention is described as following.
Within the forementioned several embodiments, the 1st item is essential characteristic, and that 2nd and 3rd are preferred.
The said ox adrenal medulla pheochromocyte (BCC) of present invention refers to the ox adrenal medulla cells capable of being dyed with dye containing chromium, which can secrete monamine, enkephalin (including monamine enkephalin (MEK), leucineenkephalin) and neurotrophy factors et al, these substances have analgesic effect.
The methods of acquiring BCC and its purification methods belong to conventional technology. For example, make ox adrenal react with collagenase to decompose collagen tissue, then seperate ox adrenal tissue into single cell by mechanical method, then filter by filter screen of 170 mesh (88 xcexcm), ox adrenal medulla cell (which contains pheochromocyte, endotheliocyte, mechanocyte and blood cells) pass through the filter screen centrifuge the fluid under the screen and remove supernatant, thus acquire the aforesaid ox adrenal medulla cell sediment, in which the pheochromocyte is about 50-60% of total cells, both endotheliocyte and mechanocyte are about 40xcx9c50% of total cells, since the shape of blood cell is small, so it usually can""t be counted in the total cells. Notably, as the BCC is about 50xcx9c60% of ox adrenal medulla cell, so which can be applied to the present invention without being purified. However, in order to improve the curing effect, it is preferably purified to a purity of 80%. The purification methods, for example, conventional wall-attaching purification method which is based on the distinct attachment tendency of different cells can be used. For example, culture mixture which contains pheochromocyte, endotheliocyte, fibroblast and blood cells are cultured in culture-flask for hours, most of fibroblasts attach the wall, while pheochromocytes, endotheliocytes and blood cells don""t attach to wall, thus most fibroblasts can be removed through changing bottle. For the blood cells can""t grow on wall, so most of blood cells can be removed through culturing them for longer time (for example 24-28 hours) and changing bottles again after the pheochromocytes and endotheliocytes being attached to wall. After changing bottle 2 times, the purity of BCC can arrive at 80% of total cells (except the blood-cell). As for the methods of cell counting, conventional counting process of observing under microscope after dyed can be used. Such as xe2x80x9cTrypan blue stainxe2x80x9d, see xe2x80x9cTissue Culture Media and Reagentsxe2x80x9d, page 1566.
In the six steps of essential technology characteristics in the present invention, the said liquid amount of each step is determined according to the confined cells number. For example, 1 liter suspension derived from step (1) of embodiment 1 contains 0.1xc3x971010 cells, while 1 liter mixture liquid from step (2) contain 0.1xc3x97108 cells, that is to say, cell concentration of suspension from step (1) is about 100 times of that from step (2), thus, it preferably disperse 1 ml suspension from step (1) into 100 ml calcium chloride solution of step (2), and the like.
The aim of forming calcium alginate bead in step (2)is to create a condition for gaining microcapsules containing ox adrenal medulla pheochromocyte in step (5). In step (5), sodium ion of sodium citrate replace the calcium ion of calcium alginate bead to form many microcapsules with small hole. Those microcapsules contain a great deal of BCC, and BCC encapsulated by sodium alginate.
There has no specific limit to the methods of forming calcium alginate bead, it only required that the sodium alginate suspension containing BCC can disperse into the solution of calcium chloride in adequately tiny liquid-drop. The diameter of said microdrop of suspension of sodium alginate is usually in the scope of 150-1000 xcexcm, preferably in the scope of 180-500 xcexcm. If micro-drop diameter is more than 1000 xcexcm, the said acquired microcapsules is too large so that it burst easily when being injected into animal or human body, that isn""t expected. Liquid-drop scatter methods usually include pinhead injection method, nebulization and so on, and nebulization is preferred, the most preferred method is to spray by electrostatic droplet generator manufactured by Toronto University, Canada (see SUN, A.M. Micro-encapsulation of pancreatic islet cell: a bio-artificial endocrine pancreas In methods in Enzymology, Vol. 137, page 575-580, 1988).
The embodiments of the present invention have been explained. After reading them and the following examples, the skill in art can comprehend the present invention easily.
Compared to the similar technology in the art, the present invention have following positive effects:
The animal experiment indicated that to inject the microcapsule containing 0.2xcx9c1xc3x97105BCC cells into subarachnoid space of spinal cord of normal rat through APA microencapsulated animal cell medicine (microcapsule diameter is 150xcx9c1000 xcexcm) can rise heat-pain threshold value (by conventional uplift-foot test and swing-tail test to detect the rat""s response to acute nocuity hot stimulation) obviously(rised 80xcx9c110%), action time last over 9 months. Clinical trials provided the evidence that implantation of microencapsulated BCC (2xcx9c9xc3x97106 cells) into subarachnoid space of spinal cord of cancer patient can relief the pain within 4xcx9c24 hours, wherein most of patients no more need to use other analgesic medicines. The pain score value (graded by two doctors according to international VAS grading method) descend obviously, spirit and appetite conditions have an improvement, therapeutic effect has last over 120 days, and it has no obvious side effect. The fact indicated that implantation of APA microencapsulated BCC can provide obvious analgesic effect, and APA microcapsule can protect implant (BCC) from being destroyed by host immune system, thus the microencapsulated BCC can survive chronically in heterogeneous animal body and act lasting analgesic effect.
The present invention have following advantages comparing to current technologies:
1. APA microcapsules according to the present invention have a small volume, which thus have at least three advantages, (1) it accelerate scattering of nutrients and metabolites, the intracapsular cells can survive chronically; (2) it is not necessary to implant hollow fiber tube with operation, but to inject microcapsules into subarachnoid space of spinal cord only by ordinary waist-penetratting method, thus it have tiny damage to tissue; (3) it usually not readily stimulate and oppress spinal cord nerve in subarachnoid space of spinal cord;
2. Compared to immune isolation membranes made of other material, APA microcapsules have proper biocompatibility;
3. Lots of experiments have indicated that APA microcapsules of the present invention have good immunity protection.
4. The BCC can secrete analgesic substances continually (last over 3 months) in host body to act sustaining analgesic effect, thus it resolved the undulatory analgesic effect resulted by clinical administration;
5. Microencapsulated BCC can act sustaining analgesic effect in host body, that avoid drug resistance and habituation conferred by repeatly using of analgesic;
6. Compared to human adrenal medulla pheochromocyte, the ox adrenal medulla pheochromocyte can be gained in gross;
7. Low temperature technology can be adopted to conserve microencapsulated BCC, which contributes to long-range transport and volume production.
In summary, the present invention has the characteristics of outstanding effect for treating pain, long acting time, safely use, convenient operation, stable quality, volume production and short production cycle. Which have wide foreground in clinic.
The following examples, experiments and application examples further explain the present invention, but they can""t be construed as limitation of the invention.